Binding assay for the detection of mycobacteria

ABSTRACT

A method for detecting the presence of Mycobacteria in a fluid or tissue which comprises mixing the fluid or tissue containing a secretory product of Mycobacteria with a complex of a tracer-containing molecule and a binding macromolecule having reversible binding affinity for the tracer-containing molecule and detecting the tracer-containing molecule, wherein the tracer-containing molecule is a charcoal-adsorbable protein from Mycobacterium tuberculosis which has a molecular weight of 20,000-30,000 and which is immunochemically stable from 4° C. to 250° C. and has a pH range from 3.0 to 9.0. The method is particularly applicable to the detection of infectious tuberculosis in humans and determining the antibiotic sensitivity of infecting Mycobacteria.

This is a division, of application Ser. No. 146,294 filed May 5, 1980U.S. Pat. No. 4,410,660.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a binding assay for the detection ofMycobacteria in fluids. In a preferred embodiment, the invention relatesto a method for the diagnosis for tuberculosis in humans.

2. Description of the Prior Art

Mycobacteria cause a wide variety of infections both in human andnon-human animals. For example, M. tuberculosis is the causativeorganism of human tuberculosis; it was isolated and identified in 1882.M. leprae is the cause of Hansen's disease and M. lepraemurium is thecause of a leprosy-like disease in rats. M. bovis is the cause of cattleinfections, and it, like M. tuberculosis also causes infections in man.M. avium is strongly pathogenic for fowl, yet not pathogenic for man.Other tubercle bacilli of the Mycobacterium genus are the so-calledcold-blooded animal type and the saprophytic types. The latter two arenot pathogenic for man.

M. tuberculosis is an almost exclusive parasite of man. It isresponsible for over 90% of all cases of tuberculosis, whereas thebovine type produces tuberculosis in man through ingestion of infectedbeef, or milk from an infected cow. Although infection from M. bovis,generally manifested as tuberculosis of the bones or lymphatic system,has been largely eliminated as a source of human infection in the UnitedStates as a result of Governmental inspection of meats, infection due toM. tuberculosis is still a major world health problem. According to theWorld Health Organization, there were 15 to 20 million infectious casesof tuberculosis in the world in 1967. The report for 1967 also statedthat two to three million deaths due to tuberculosis occur each yearwith 80% of the deaths in developing countries. (Pelczar, Jr. and Reid,"Microbiology", Third Edition, McGraw-Hill (1972), p. 78). In 1976, morethan 32,000 new cases were reported in the United States alone.Eradication of tuberculosis in man and similar infections in non-humananimals is therefore still of utmost significance in the United Statesand the rest of the world.

Detection of actively growing Mycobacteria in human and non-humananimals has been carried out in the prior art by classical staining andculture methodology. Mycobacteria are difficult to stain with the usualmicrobiological dyes, but they stain readily by the Ziehl-Neelsentechnique (initial staining with basic fuchsin washed with acid andalcohol). Probably because of the high fat content of the organisms,they are not decolorized by the acid-alcohol and therefore have beentermed acid-fast organisms. The requirement for isolation and culture ofMycobacteria from relatively inaccessible organs coupled with the slowmultiplication rate of the tubercle bacillus, presents a problem forrapid diagnosis. In fact, since tuberculosis is a chronic baterialdisease, advancing slowly, the primary infection may go unnoticed untila chance x-ray reveals lung lesions.

The demonstration of tubercle bacilli in body discharges--sputum,gastric contents, spinal fluid, urine, etc.--is the final proof incorroboration of clinical diagnosis. Laboratory methods includemicroscope examination of stained samples for the presence of bacilli,planting the suspected material on suitable culture media, and animalinnoculation with concentrated sputum or other material in which theorganisms may be found.

Another diagnostic method is the use of the tuberculin test. The test isperformed by injecting intracutaneously small amunts of tuberculin, apurified protein derivative from cultured tuberculosis bacilli. Apositive test is indicated by an inflammatory reaction at the site ofinjection within 48 hours. A positive tuberculin test, however, is notnecessarily an indication of an existing infection in adults for it maysimply mean that they once harbored the bacilli or some non-pathogenicMycobacterium or an atypical M. tuberculosis. In children, a positivetest is usually an indication for treatment. X-ray examination isgenerally employed to corroborate pulmonary infection. However, atpresent only identification of Mycobacteria in cultures of clinicalspecimens can be considered proof of active disease. Alternative andsomewhat more efficient methods for rapid detection of growingMycobacteria, especially for the rapid diagnosis for tuberculosis, havebeen proposed in the prior art. Thus, Odham et al (J. Clin. Invest.63:813-819 (1979)), have demonstrated the presence of tuberculostearicacid in sputum from patients with pulmonary tuberculosis by selectiveion monitoring.

Straus and Yalow (Clinical Research, Volume 25, No. 3, April 1977, A384)describe a radioimmunoassay (RIA) procedure for the detection oftuberculin purified protein derivative (PPD) shed into culture media bygrowing Mycobacterium tuberculosis. In this procedure, antisera wereraised in guinea pigs by repeated subcutaneous injection of PPD (areadily available commercial product). PPD was iodinated with ¹²⁵ I andanalysis of the radiolabeled PPD by Sepharose 6B column chromatographyyielded a peak of radioactivity in the void volume (VVP) which was usedas a tracer in RIA. Separation of the antibody-bound and free tracer wasachieved by precipitation with rabbit anti-guinea pig γ-globulin. Thisradioimmunoassay is not useful, however, for the detection of M.tuberculosis or any other Mycobacterial species which are activelygrowing in an animal host such as a human or non-human infected animal.The Straus and Yalow assay (as described in the last two lines of theAbstract) was not sensitive enough to be employed for the rapid anddirect identification of M. tuberculosis in biological fluids. The voidvolume peak (VVP) is generally too unstable to provide suitably labeledantigens and has the further disadvantage that precipitation withanti-guinea pig globulin is necessary to separate free from boundlabeled antigens, since the VVP does not absorb on to charcoal.

A need, therefore, exists for a highly sensitive, rapid and efficientmethod for the detection of actively growing Mycobacteria in human andnon-human animals. More particularly, a need exists for a rapiddiagnostic method for the many different forms of tuberculosis inhumans.

SUMMARY OF THE INVENTION

It is therefore an object of this invention to provide a method for thedetection of actively growing Mycobacteria in human and non-humananimals.

It is another object of the invention to provide a diagnostic method fortuberculosis in humans.

Still another object of the invention is to provide a binding assaymethod for the detection of Mycobacteria in animals by detectingsecretory products of Mycobacteria in the biologic fluids of saidanimals.

Other objects of the invention are to provide a method for studyingMycobacterial growth and to provide a method for determining thesensitivity of Mycobacteria to the action of antibiotics.

Yet another object of the invention is to provide a radiolabeledtuberculoprotein derived from Mycobacterium tuberculosis which is heatstable and charcoal-adsorbable.

A further object of the invention is to provide a process for thepreparation of the aforementioned radiolabeled tuberculin purifiedprotein derivative.

Still a further object of the invention is to provide a diagnostic kituseful for the diagnosis by binding assay methodology of tuberculosis inhumans.

These and other objects of the invention, as will hereinafter becomemore readily apparent, have been attained by providing:

A method for detecting the presence of Mycobacteria in a fluid or tissuewhich comprises:

mixing said fluid or tissue, containing a secretory product ofMycobacteria with a complex of a binding macromolecule and atracer-containing molecule, and

detecting said tracer-containing molecule;

wherein said tracer-containing molecule is a heat stable,charcoal-adsorbable protein purified from Mycobacterium tuberculosiswhich has a molecular weight of 20,000-30,000; preferably 24,000Daltons.

Another object of the invention has been attained by providing acharcoal-adsorbable protein purified from Mycobacterium tuberculosiswith a MW of 20,000-30,000, preferably 24,000 Daltons, which isimmunochemically stable from 4° C. to 250° C. and over a pH range from3.0 to 9.0, and which has well-defined mobility characteristics onstarch gel electrophoresis.

Still another object of the invention has been attained by providing amethod of preparing a tracer-containing protein purified fromMycobacterium tuberculosis which comprises:

labeling a mixture of tuberculoproteins isolated from the growth mediaof Mycobacterium tuberculosis;

purifying from said mixture of labeled tuberculoproteins a labeledheat-stable, charcoal-adsorbable protein having a molecular weight of20,000-30,000, preferably 24,000 Daltons.

Yet another object of the invention has been attained by providing amethod of preparing a tracer-containing protein as describedhereinbefore, wherein the purification is carried out by

chromatographing a mixture of labeled tuberculoproteins isolated fromthe growth media of M. tuberculosis on beads of dextran cross-linkedwith epichlorohydrin, having a fractionation range for peptides of3,000-80,000 and

fractionating from said chromatographic step a purified, heat-stable,charcoal-adsorbable protein having a MW of 20,000-30,000.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 shows the gel chromatography of ¹²⁵ I tuberculoproteins on aSephacryl S200SF column. The most immunoreactive fraction is denoted as(A). See Example 1;

FIG. 2 shows the rechromatography of fraction (A) from FIG. 1, on aSepharose 6B column. The most immunoreactive fraction is denoted as (B).See Example 1;

FIG. 3 shows the rechromatography of fraction (B) from FIG. 2 on aSephadex G75. The most immunoreactive fraction is denoted as (C);

FIG. 4 shows a standard curve for the radioimmunoassay of thetuberculoprotein. Dilutions of autoclaved PPD and autoclaved culturemedium are superimposable on the dilution curve of authentic standard;see Example 1.

FIG. 5 shows the starch gel electrophoresis of the purified protein fromM. tuberculosis having a MW 24,000. Conditions:

Borate Buffer, pH 8.6, 80 volts, 12 hours. Legend: Alb: albumin; BpB:Bromophenol Blue.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention has succeeded in providing a highly sensitive,fast, safe and effective method for the detection of Mycobacteria in thebiological fluids of human and non-human animals. The present inventorshave discovered that when Mycobacteria infect an animal host, theMycobacteria secrete, into the physiological fluids of said host,tuberculoproteins which can be detected by a highly specific bindingassay. This is the first time that the presence of an infectingmicroorganism has been detected by measuring the amount of a secretoryproduct of said microorganism in the biological fluids of the host. Itis quite likely that all metabolically active bacteria secrete non-toxicspecies and strain-specific products. However, until this invention, theprimary identification of such materials has not commonly been exploitedas a diagnostic method. The development of a binding assay for aMycobacterial secretory product thus represents a new diagnosticapproach.

The essence of the invention relates to the preparation of a stable,tracer-containing protein from M. tuberculosis capable of being used ina binding assay for the secretory products of Mycobacteria. This stable,tracer-containing protein is prepared from a readily availabletuberculin purified protein derivative (PPD) obtainable from ConnaughtLaboratories Limited, Toronto, Ontario, Canada. The preparation of PPDis fully described in Gupta and Landi (Canadian Journal of Microbiology,24: 1242-1249 (1978)) and Landi, S., (Appl, Microbiology, 11: 408-412,1963). The description and preparation of PPD has also been published inLandi, S. et al in: Proceedings of Third Conference of Evaluation ofProcedures for Tuberculin Testing, U.S. Dept. of HEW; Atlanta, Ga., May4, 1971 (39-47), and Landi, S., et al, Tubercle, 59: 121-133 (1978). Anyacid or salt precipitated mixture of tubercular proteins isolated fromthe growth media of actively growing Mycobacteria can be used as thestarting material for the preparation of the tracer-containing molecule.PPD is but one of the preferred examples of such mixture of tubercularproteins.

The mixture of tubercular proteins, preferably PPD, is labeled in orderto prepare the tracer-containing molecule. Any label useful in bindingassays, particularly in radioimmunoassays, can be used. Tracer moleculescan be divided into two types: those with an internal label and thosewith an external label. With an internal label, an existing atom in theligand molecule is replaced by a radioactive isotope of that atom (e.g.,C¹⁴ for C¹², H³ for H¹). With an external label, an atom or atoms of aradioactive isotope (e.g., I¹³¹ or I¹²⁵) are substituted for an existingatom on the ligand molecule; to achieve stability, a covalent link isestablished between label and ligand. A tracer with an external labelsuch as I¹²⁵ is not identical with the unlabeled ligand, but itsbehavior is practically indistinguishable from the latter. Both theinternal or external label methodology can be used in the presentinvention. The most preferred method is that of using an external label,most preferably radioactive iodine. It is known that iodine can besubstituted into the aromatic side-chain of tyrosine residues, as wellas other amino acids such as histidine. Many procedures have beendescribed for iodination, and they can all be used in the presentinvention. The following methods of iodination have been described:

1. Chloramine T technique (Greenwood, F. C., et al, Biochemical Journal,89, 114 (1963)). The procedure is simple since all that is required ismixing a solution of the protein mixture, sodium iodide havingradioactive iodine and chloramine T; the reaction is terminated by theaddition of a reducing agent, preferably sodium metabisulfite.

2. Iodine monochloride technique (McFarlane, A. S., Nature 182, 53(1958)). A solution of iodine monochloride is mixed with solutions ofthe radioactive isotopes and the protein mixture.

3. Chlorine and hypochloride technique (Redshaw et al, Journal ofEndocrinology, Vol. 60, 527 (1974)).

4. Lactoperoxidase (Marchlonis, J. J., Biochemical Journal, 113, 299(1969)). In this technique enzymatic iodination using lactoperoxidase inthe presence of a trace of hydrogen peroxide is carried out on theprotein mixture. The advantage is that the protein mixture is notexposed to high concentrations of a chemical oxidizing agent such aschloramine T. Furthermore, a reducing agent is not required since simpledilution will stop the reaction. Alternatively, the lactoperoxidase canbe attached to a solid phase and removed by centrifugation. Thepreparation of reagents and conditions for the reaction itself are moretechnically demanding than those for the chloramine T procedure.

5. Electrolysis (Rosa, U., et al, Biochem. Biophys., Acta, 86, 519,(1964)). This technique involves the use of iodide in the reactionmixture being converted to reactive forms by the passage of an electriccurrent.

6. Iodine vaporization. A mixture of chloramine T and isotopic sodiumiodide yields gaseous iodine. The reaction is carried out in a gas-tightouter vessel in which is a smaller inner vessel containing a solution ofa protein mixture; the reactive iodine vapor diffuses into the solution.

7. Conjugation labeling technique (Bolton, A. E., et al, BiochemicalJournal, 133, 529 (1973)). In this procedure the iodine is first coupledto an appropriate carrier "handle" containing a phenol or imidazolegroup for iodination, and an amine for coupling to the ligand or itsderivative.

For a general description of all of these labeling techniques see Chard:"An Introduction to Radioimmunoassay and Related Techniques", NorthHolland Publishing Company, Amsterdam, N.Y., Oxford, First Edition,1978.

Labels other than radioactive labels can, of course, be used since theyare well known in generalized binding assay techniques. Thus, forexample, alternatives to isotopic labels may be: (a) chromophoriclabels, such as fluorescent, ultraviolet-absorbing or visiblelight-absorbing labels: These are advantageous because of their longshelf life and absence of radiation effects; (b) Enzyme labels(Enzyme-linked immunoabsorbent assay ("Elisa")): Since specific enzymescan be coupled to other molecules by covalent links, a highly specificenzyme may be covalently reacted with the partially purifiedMycobacterial protein mixture and the resulting "tracer-containingmolecule" can be purified to obtain the required fraction for use in thebinding assay (see Chard, supra, at 374-375); alternatively, pureprotein can be linked to enzyme (see Engvall et al, J. Immunol. 109, 120(1972)); (c) other tracers such as free radical labels or bacterialphase labels could also be used in the present invention (see Chard,supra, at 376). The most preferred method of preparing atracer-containing molecule is radiolabeling with radioactive iodine.

Once the labeled protein mixture has been prepared by any of theaforementioned techniques, the desired tracer-containing molecule ispurified therefrom, by an appropriate purification method, includingchromatography. Two gel filtration chromatographic techniques and asilica gel technique can be described here as examples:

1. One-step Gel Filtration Technique. The labeled mixture isfractionated on a chromatographic column containing beads of dextrancross-linked with epichlorohydrin (known as Sephadex) and having afractionation range for peptides of 3,000-80,000. After elution in anappropriate buffer and fractionation, the peak containing a proteinfraction having a molecular weight of 20,000-30,000 preferably about24,000 Daltons is isolated and utilized. The tracer-containing purifiedprotein from M. tuberculosis thus isolated easily adsorbs on charcoal,and shows a high degree of stability. It shows a remarkable degree ofheat stability, remaining immunochemically intact over a range of 4°C.-250° C. This can be ascertained by demonstrating that an autoclavingtreatment at 250° C. does not change the immunochemical specificity ofthe protein (see FIG. 4). The protein also shows a high degree of acidstability, being immunochemically unchanged over the range of pH3.0-9.0. It has a shelf life of several months, at pH 8.0-9.0, 4°C.-room temperature.

The final product can also be characterized by its characteristicbehavior on starch gel electrophoresis. Using this electrophoretictechnique in borate buffer, pH 8.6 at 80 volts for 12 hours, the ¹²⁵I-tracer-containing molecule comigrates with bovine serum albumin.

In contrast, the VVP material described by Straus and Yalow (ClinicalResearch, Vol. 25, No. 3, A384, 1977) is non-charcoal adsorbable, has adifferent gel electrophoresis pattern, and is only stable for a fewhours. In addition, since it is isolated from the void volume peak of aSepharose 6B column, the molecular weight is in the millions, and notabout 24,000 as the protein of the present invention. The materials aretherefore very different.

2. Three-stage Gel Filtration Technique. In this purification, thelabeled protein mixture prepared above, is first fractionated on a gelpermeation chromatographic column of allyl dextran cross-linked withN,N'-methylene-bis-acrylamide, having a MW fractionation range forproteins of 5×10³ -2.5×10⁵. This material is commercially known asSephacryl S200. The most immunoreactive peak is isolated andrefractionated on a gel chromatographic column of neutral agarose beads,which is substantially free of charged groups, and has a proteinfractionation range of 10⁴ -4×10⁶. This material is commerciallyavailable and known as Sepharose 6B. The refractionated material havingthe highest immunoreactivity is thirdly added to a gel chromatographiccolumn of dextran beads crosslinked with epichlorohydrin (Sephadex G75,supra), having a MW fractionation range for proteins of 3,000-80,000.The material having a molecular weight of 20,000-30,000, preferably24,000 Daltons, which is heat stable and charcoal-adsorbable is isolatedand used. This material is essentially similar to the material purifiedby the one-step technique.

In both the one-step and three-step techniques, the appearance ofpurified protein can be readily followed by the use of the incorporatedlabel.

3. Silica Gel Technique. Purification or repurification can also beperformed by adsorption and elution from silica gel, where adsorption iscarried out at pH 1.0-4.0, preferably 2.0-3.0, and elution at pH7.0-9.0, preferably 8.0-9.0.

The aforementioned three chromatographic techniques, as well as anyother purification techniques, can be operated in any buffer normallyused therefor. Examples of such buffers are phosphate, borate, tris,veronal, glycine, histidine, PIPES, HEPES, and the like, atconcentrations depending on the desired buffer capacity, easilydeterminable by one skilled in the art, normally 0.01M-1.0M.

The tracer or label-containing purified Mycobacterial constituent thusprepared, can be used successfully in any of the well known competitivebinding assays, of which radioimmunoassay is but one example. With thisstable, charcoal-adsorbable tubercular protein from M. tuberculosis astracer, it is possible for the first time to detect the presence ofMycobacteria, by detecting minute amounts of Mycobacterial secretoryproducts which are shed into fluids or tissues. In order to carry out acompetitive binding assay, preferably a radioimmunoassay, it isnecessary to provide a binding macromolecule which has reversibleaffinity for the tracer or label-containing molecule, and forMycobacterial secretory products. Such a binding macromolecule is mostgenerally an antibody raised against the Mycobacterial secretoryproduct. It is also obvious that the binding macromolecule, preferablyantibody, should as far as possible be directed only to theMycobacterial secretory proteins which the assay is intended to measure,not to a wide variety of similar materials which would interfere withspecificity.

The preparation of anti-sera in animals is a well-known technique (seefor example Chard, supra, pp. 385-396). The choice of animal is usuallydetermined by a balance between the facilities available, and the likelyrequirements in terms of volume of the resulting antiserum. A largespecies such as goat, donkey and horse may be preferred, because of thelarger volumes of serum readily obtained, however, it is also possibleto use smaller species such as rabbits or guinea pigs which often havehigh titer antisera. Usually, subcutaneous injection of MycobacterialPPD coupled to guinea pig albumin and emulsified in Freund's completeadjuvant is sufficient to produce anti-PPD antisera. The detection ofappropriate antibodies can be carried out by testing the antisera withappropriately labeled tracer-containing molecules. Fractions that bindtracer-containing molecules are then isolated and further purified ifnecessary.

It is of course possible to use other than antibodies as the bindingmacromolecule. The use of cell receptors specific for Mycobacterialproteins, or of any circulating binding proteins equally specific forMycobacterial proteins can be used.

The general competitive binding assay techniques useful for thedetection of minute amounts of organic molecules such as hormones,proteins, antibodies, and the like are well known in the art (see Chard,supra). Any of these competitive binding assay techniques can be usedfor the purposes of the present invention.

A small amount of the fluid or tissue sample suspected of containing theMycobacterial secretory product is incubated with a complex of thelabeled tracer-containing purified constituent and the bindingmacromolecule therefor, preferably an antibody raised againstMycobacterial proteins. Any fluid or tissue sample derived from a sourcesuspected of containing Mycobacteria can be used. This includes thephysiological fluids of human and non-human animals, such as urine,blood, spinal fluid, and the like.

Apart from humans, the most common animals which are subject toMycobacterial infections are bovines, fowl and rabbits. However, othermedia may be contaminated with Mycobacteria or their secretory products:these might include liquid sources such as suspected contaminated milk,water or other consumable or non-consumable liquids. All that isrequired of the liquid or tissue being examined is that it be capable ofcontaining Mycobacterial secretory products shed thereinto.

Once the incubation of the test sample with the complex of the bindingmacromolecule and tracer-containing molecule is complete, it isnecessary to determine the distribution of the tracer-containingmolecule between the free and the bound form. Usually, but not always,this requires that the bound fraction be physically separated from thefree fraction; a variety of techniques can be used for that purpose. Allof the techniques exploit physical-chemical differences between thetracer-containing molecule in its free and bound form. The generalavailable methodologies have been described by Yalow (Pharmacol. Rev.28, 161 (1973)). These techniques include adsorption of free antigen tosolid phase material, such as cellulose, charcoal, silicates or ionexchange resins; precipitation of antigen-antibody complexes by secondantibody; salting out techniques or organic solvents; adsorption orcomplexing of antibody to solid phase material; electrophoreticseparation on cellulose, starch gel or polyacrylamide gel, and the like.(See also Chard, supra, pp. 405-422). Suitable organic solvents includeethanol, dioxane, polyethylene glycol, trichloroacetic acid, a solutionof sodium sulfite, and a solution of ammonium sulfate.

The choice of a technique depends on the speed, simplicity,applicability and cost. It is a simple matter of choice for anyoneskilled in the art and therefore the generalized techniques will not bedescribed in further detail.

Particularly preferred among the aforementioned techniques areadsorption methods, double-antibody methods and solid phase systems.

In adsorption methodology, the non-specific adsorption of proteins toparticle surfaces is used as a method for the separation of bound andfree tracer-containing molecules. This procedure depends on the factthat only the tracer-containing molecules and not the bindingmacromolecules or bound complexes have the adsorption property. The mostpreferred adsorption procedure which is highly useful in the presentcase, is adsorption on charcoal or silicates. The most commonly used ofthe available charcoals are the Norit range (Norit SXl) with a maximumparticle size of 63 μm. Considerable batch-to-batch variation may befound with these and it should never be assumed that the material inbottles with apparently identical labels will behave identically in anyassay. Each batch, therefore, has to be carefully tested before it isput into routine use. Particular silicates have adsorptive propertieswhich advantage is taken for the separation. Materials employed mayinclude talc, microfine precipitated silica (Q_(uso)) and Fuller'searth.

"Double" or "second" antibody methods depend on the precipitation of thebound complex with an antibody directed to the macromolecule. The secondantibody is specific to the γ-globulin of the species in which the firstantibody was raised, for example if a guinea pig anti-PPD serum is usedin the primary reaction of an assay for Mycobacterial secretoryproducts, an atiserum to guinea pig γ-globulin raised in a goat may beused for the separation step. Although most commonly used inradioimmunoassays, this concept can be applied to any bindingmacromolecule for which an antiserum is available. Separation by thistechnique requires a relatively large concentration of second antibodyand a correspondingly large amount of the species of γ-globulins ofwhich the first antibody forms a part must be included; for thispurpose, a second antibody system always involves the addition ofcarrier protein, either whole serum or γ-globulin from the species inwhich the first antibody was raised. The use of second-antibodytechniques suffers from two important practical disadvantages. The firstis that it requires an additional period of incubation which may rangefrom one to twenty-four hours and can, therefore, considerably extendthe time required to complete the assay. A second practical disadvantageis that of reagent supply. A new second antibody requires carefulevaluation and of those tested few will turn out to be completelysatisfactory. Relatively high concentrations are required and theproduct of one animal is only sufficient for a limited number of assays.Second-antibody systems are therefore also quite expensive. The use ofcoupling of the second antibody to an insoluble matrix such as celluloseis more economical and efficient and has been described by den Hollanderet al. (Kirkham et al "Radioimmunoassay Methods" (1971) p. 419).

Solid phase systems in general have been increasingly utilized in recentyears. When the binding macromolecule is covalently coupled to aninsoluble support, then both it and the bound complex can readily beseparated from the soluble free fraction. A wide variety of solid-phasesupports have been described, which include particles of dextran andcellulose, and continuous surfaces such as polystyrene or polypropylenediscs, or the walls of plastic or glass tubes. Plastic surfaces exhibitadsorptive properties, and simply exposing such surface to anappropriate dilution of the anti-PPD antiserum will lead to theattachment of a proportion of the antibody molecules thereon. The bondis probably ionic or hydrophobic and not covalent. Covalent bonding,however, can be readily obtained by the incorporation of cross-linkingagents such as glutaraldehyde and other agents in the antibody solutionused for the coating. Coated tube systems offer great convenience in theactual performance of assays and the technique can be widely use incommercial kits. In one preferred embodiment, the anti-PPD antibody iscovalently attached to the inside of a test tube and tracer-containingmolecule is also incorporated in the tube. A single addition of a sampleof fluid or solution of tissue being tested is then added to the testtube. After incubation, the contents of the tube are emptied and thetracer is detected by standard methodology.

The binding macromolecule can also be attached to a particulate solidphase by any one of a number of techniques designed to yield a covalentlink between the protein and the particles, such as for examplediazotization or cyanogen bromide activation. The resulting material isthen extensively washed to ensure that no free γ-globulin moleculesremain. Alternative approaches include the use of antibody entrapped inthe interstices of a polyacrylamide gel or covalently bound to magneticparticles (polymer-coated iron oxide). With the latter system, mixingand separation can be simply achieved by the application of a magneticfield.

An alternative binding approach to the detection of Mycobacterialsecretory product from fluids or tissues is by using the recentlydeveloped latex particle agglutination technique. This technique doesnot involve the use of a tracer or label-containing ligand havingreversible affinity for the binding macromolecule but rather the use ofthe unlabeled ligand itself. See for example, Sawai et al, U.S. Pat. No.4,118,192 or Hoffmann-LaRoche, British Pat. No. 1,384,399. Thetechniques described in these two patents are readily applicable to thespecific process of the present invention. In these techniques, antibodyraised against PPD is supported on an insoluble carrier particle,usually a latex particle, thus sensitizing the insoluble carrierparticle. The supported anti-PPD is then reacted with the samplesuspected of containing the Mycobacterial secretory tubercular proteinproduct. The sensitized latex agglutinates to a degree which isproportional to the amount of secretory product present in the fluid ortissue. The agglutination is then followed by irradiating the resultingreaction mixture with light having a wave-length in the range of 0.6-2.4microns. The determination of absorbance can be performed with aspectrophotometer similar to that used in near infrared spectrometry.Polystyrene latexes or styrene butadiene-latexes can readily be used;however, other particles such as dispersed coccal bacteria, cellmembrane fragments, microparticles of inorganic oxides such as silica,silica alumina and alumina or finely pulverized minerals, metals and thelike are also readily useable. These latex agglutination techniques notonly make it possible to determine low concentrations of Mycobacterialsecretory product, but enables the determination of such secretoryproducts in trace amounts and with comparable specificity to those ofthe radioimmunoassay methodology. The amount of secretory Mycobacterialproduct can be determined by measuring the absorbance as describedabove, or alternatively by measuring the rate of reaction, or thereaction time required for the absorbance to reach a prescribed value.The Sawai et al methodology is also applicable in theinhibition-of-agglutination mode. In this mode, latex particles arecoated with protein purified from M. tuberculosis having a molecularweight of about 24,000, which preparation has been extensively discussedabove. These particles are then incubated with anti-PPD. The so-formedcomplex is mixed with test fluid or tissue suspected of containingMycobacterial secretory products. If the test sample contains secretoryproducts, the latter will compete for the antibody binding sites andinhibit the agglutination of the antigen-covered latex particles. Theprotein of molecular weight 24,000 used for the radio-binding assaysdescribed previously need not, in the latex techniques, contain anyradioactive label or tracer. It may be useful, however, for simplepurposes of purification and isolation of this protein, to proceed asindicated previously and incorporate a label of tracer.

When radioimmunoassay is utilized as the mode of detection ofMycobacterial secretory products, and after incubation of the testsample with the antibody-tracer-containing molecule complex andseparation of the tracer-containing molecule, it is necessary to detectthe tracer by some physical or chemical means. When the tracer is thepreferred radioactive iodine, scintillation counting is the method ofchoice. Radioactive iodine is a γ-ray emitter and therefore intimatecontact between the isotope and the scintillator is unnecessary. Thescintillator in these cases usually consists of a crystal of sodiumiodide coated with thallium, usually formed as a well; as the radiationstrikes the molecules making up the crystal lattice, ionization occursand results in a light flash which is then detected by the photomultiplier. If the radioisotope used for labeling is C¹⁴ or H³, liquidscintillation is appropriate to detect β-particles.

The data obtained from tracer binding assays can be plotted in any of avariety of standard plots. A commonly used method is a plot of percentbound, or the ratio of bound to free ligand (B/R) as a function of thestandard concentrations of secretory product. The inverse ratio of freeto bound (F/B) can also be used. Many workers employ a semilogarithmicplot of percentage tracer bound against the log of concentration ofunlabeled secretory product. The choice of plot is very much a matter ofpersonal taste or experience of the individual workers. Generally, it isnecessary to prepare a series of standards containing differentconcentrations of secretory protein, such as PPD. A standard curve isthen prepared which can be used for any subsequent radioimmunoassaydeterminations. The validity of the assay is independent of the choiceof plot employed.

The methodology of the present invention can be used to detect a widevariety of Mycobacterial species. Although material derived from M.tuberculosis reacts most strongly, there is wide cross-reactivity withother pathologic Mycobacterial species. Immunoreactive material can bedetected in culture media and test samples of all disease producingMycobacterial species and no reactivity is detected in media or testsamples from diverse bacterial or fungal species. The cross-reactivityobserved with the assay is consistent with the amino acid compositionsof tubercular proteins from a variety of Mycobacterial species, whichcompositions have recently been found to be very similar (Landi, S. etal, Ann. Sclavo 13, 862-883 (1973)). This pattern of cross-reactivityappears favorable since it allows identification of Mycobacterialinfection caused by a variety of species. The efficacy of the method canalso be enhanced by working with contaminated specimens containinggreater numbers of Mycobacteria. The availability of tubercular proteinderived from other species offers the possibility of developingadditional assays for greater diagnostic specificity. In this respect,it is possible to carry out a preliminary screening for the generalpresence of Mycobacteria in the test sample, followed by a more specificdetermination for the type of species of Mycobacteria in the testsample. If species identification were desired, the sample could beassayed in several similar assay systems each employing antisera withenhanced specificity for a given species of Mycobacteria. Among thedetectable Mycobacterial species are M. leprae, M. lepraemurium, M.tuberculosis, M. bovis, M. marinum M. avium, M. Phlei, M. smegmatis, M.intracellulare, M. simiae, M-xeuopi M. kansasii, M. batti M. ofricanium,M. ulcerans and the like.

Another very useful application of the method of the invention is in thedetermination of susceptibility of Mycobacteria to antibioticsubstances. Drugs such as isoniazid, streptomycin, ethionamide,tetracyclines, rifampin, ethambutol, aminosalicyclic acid andpyrazinamide are commonly used in the treatment of tuberculosis. A quickmethod to ascertain whether the infecting Mycobacteria is susceptible toany of these, is to determine the presence of immunoreactive secretoryproducts of bacteria which are growing in the presence of the drugs.

The techniques and materials of the present invention for the detectionof Mycobacterial secretory product in test samples or tissues can bereadily automated. A noteworthy development in the field of automatedradioimmunoassay is the recent patent of Brooker et al, U.S. Pat. No.4,022,577. Among the kits useful in the present invention are those ofthe general type described by Szczesniak, U.S. Pat. No. 3,899,298. Suchkits comprise a carrier being compartmented to receive at least one, orat least two, or at least three or more, vials and to maintain saidvials in closed confinement. A first vial may contain tracer- orlabel-containing ligand molecules such as for example the radiolabeledprotein of molecular weight 20,000-30,000, preferably 24,000 Daltons.Another vial may contain anti-PPD antibodies raised in an appropriateanimal. These materials may be in the freeze-dried state or suspended ina buffer solution. When in a freeze-dried state, the buffer solution maybe in a third vial. Alternatively, the complex of anti-PPD andtracer-containing molecule may be present in one vial and buffersolution which may be added at the time of testing may be present inanother vial. Alternatively, the first vial may be a test tube beingcovalently coated at the inner surface thereof with antibodies, e.g.,anti-PPD. A second vial may be the tracer-containing purifiedMycobacterial protein of MW 20,000-30,000, in the presence or absence ofbuffer. At the time of test, the buffer suspension of tracer-containingmolecule is added to the antibody-coated test tube and a drop or two oftest sample containing the suspected Mycobacterial secretory products isadded to the test tube. Alternatively, the first vial may be a test tubecoated at its inner surfaces with anti-PPD and containing incomplexation therewith the tracer-containing molecule. At the time oftesting, the addition of the suspect test sample is then sufficient tocarry out the methodology. Other vials in the carrier may contain theelements necessary for the separation of bound and freetracer-containing ligand. Thus, such vials may contain charcoal,silicates, or second-antibodies useful in the "second-antibodytechnique" described previously. Any number of variations andpermutations consistent with the various techniques described previouslycan be envisioned for the preparation of the kit. These are all mattersof choice determined by the ease of handling, rapidity and efficiency ofthe testing. Other apparatus useful for the present invention are forexample, the recently described "gamma stick", Schen, U.S. Pat. No.4,135,884. Schen describes a test stick adaptable to be introduced intoa test tube. The stick has a test portion adapted to be coated withantibody, in this case anti-PPD. The test tube containsantigen-containing solution. A variation of this technique is describedin Bratu, U.S. Pat. No. 3,826,619, and is also readily applicable to thepresent invention. Another useful apparatus for the present invention isthat of Updike, U.S. Pat. No. 3,970,429. Updike describes a syringe-likeapparatus loaded with hydrophilic insoluble porous gel particles havingbinding macromolecules trapped therein. These binding macromoleculeswould be, for example anti-PPD. A fluid to be tested is introduced intothe syringe by means of a plunger and contacts the gel particles wherebysome of the binding sites of the binding macromolecules are occupied.This is followed by exposure of the gel particles to radioactive taggedtracer-containing material, followed by measurement of the radioactivityof the unbound or bound tagged material. The binding proteins ormacromolecules in the gel particles are returned to the original statefor reuse by treatment with acidic medium to affect detachment of thebound material, followed by washing to affect removal of the unboundmaterial.

Having now generally described the invention, the same will be furtherillustrated by means of specific examples which are presented herewithfor purposes of illustration only and are not intended to be limitingthereof, unless otherwise specified.

EXAMPLE 1

Iodination of PPD. PPD, (Landi, S. Appl. Microb., 11, 408-412 (1963))was dissolved in 0.25M phosphate buffer pH 7.5 at a concentration of 3.5mg per ml. Iodination was done using a minor modification of achloramine T technique described in Berson, S. A. and Yalow, R. S.(General Radioimmunoassay, S. A. Berson and R. S. Yalow, "Methods inInvestigative and Diagnostic Endocrinology", Part I, NorthHolland-Publishing Company, Amsterdam, 1973, pp. 84-120). For a typicaliodination, approximately 200 MCi of ¹²⁵ I (commercially available) and1.0 μg PPD were exposed to 52 μg of chloramine T for about 3 secondsbefore the addition of 96 μg of sodium metabisulfite. About 1 μl of theiodination mixture was added to plasma and applied to paper forchromatoelectrophoresis to monitor the extent of iodination.

Three Stage Chromatographic Purification. The iodination mixture wasthen placed at the top of a 1×50 cm Sephacryl S200 SF column and elutedwith 0.02M barbital buffer, pH 8.6, containing 2% fetal bovine serum(standard diluent) at a flow rate of 10 ml per hour. A portion of each 1ml fraction was then tested for binding to antiserum as described belowunder radioimmunoassay methodology. The Sephacryl S200 SF elutionfraction which bound best to antibody was then placed at the top of a1×50 cm Sepharose 6B column and eluted as described above. The Sepharose6B elution fraction with the greatest immunoreactivity was then furtherfractionated on a 1×50 cm Sephadex G75 column and each fraction wasagain examined for binding to antibody.

During iodination, greater than 75% of the ¹²⁵ I was incorporated intoprotein. However, since the commercial PPD is quite heterogeneous, achromatographic pufification is required to obtain a fraction of ¹²⁵I-PPD with optimal properties of binding to antibody. In the originalfractionation on Sephacryl S200 SF, labeled PPD which combines toantibody is found in void volume eluates as well as in eluatescorresponding to the major peak of radioactivity (FIG. 1). Fraction A inFIG. 1, on the descending limb of the major peak of radioactivity had aB/F ratio of 0.295 when used with a 1:2000 dilution of the antiserum.After testing, it was kept frozen for six weeks at -20° C.;rechromatography on Sepharose 6B as described above (FIG. 2) was carriedout. The best fraction, B, had a B/F ratio of 0.521 when tested underthe same conditions as A. Deiodination, which had occurred during thesix weeks storage, accounts for the late eluting peak. Rechromatographyon fraction B on Sephadex G75 as described above, resulted in a majorpeak of radioactivity at about 1/4 of the way between the void volume onthe iodide peak (FIG. 3). Fraction C, on the ascending limb of the majorpeak, was the most immunoreactive (B/F ratio of 0.902). Thus labeledantigen can be stored for a period of several months and refractionatedwhen needed for assay.

Silica Gel Purification Technique. The iodination mixture was adjustedto pH 2.3 before addition of 5 mg microfine precipitated silica (QUSOG32, commercially available). After mixing and centrifugation (3,000rpm×15 min), the QUSO with adsorbed ¹²⁵ I-tuberculoprotein was washedtwice with 3.0 ml distilled water adjusted to pH 3.2 with 0.1N HCl. Thelabeled material was then eluted from QUSO in 0.05 ml barbital, pH 8.6,containing 2% fetal bovine serum.

Preparation of the Antiserum. Six guinea pigs received a subcutaneousinjection of about 1 mg of PPD coupled to guinea pig albumin bycarbodiimide and emulsified in Freund's complete adjuvant. The guineapigs were bled two weeks after immunization. Two of the six guinea pigs(GP4 and GP5) had circulating antibody which bound ¹²⁵I-tuberculoprotein. Two weeks after a second injection, antibody titerswere unchanged in GP4 and GP5. None of the other animals had significanttiters.

Radioimmunoassay. Radioimmunoassay was performed in 1.0 ml of standarddiluent containing GP5 plasma in a final concentration of 1:2000 and atracer concentration of about 0.001 μCi ¹²⁵ I-tuberculoprotein. PPDdiluted to a concentration of 10 μg/ml in a standard diluent was used asstandard. Unknown samples were generally assayed in final dilution of1:40. Incubation was carried out overnight at 4° C. Separation ofantibody bound and free ¹²⁵ I-tuberculoprotein was accomplished byaddition of 0.2 ml of uncoated charcoal (Norit A) suspended in 0.02Mbarbital, pH 8.6, at a concentration of 100 mg per ml.

Culture technique. M. tuberculosis and M. fortuitum were culturedaerobically at 35° C. in Middlebrook 7H9 medium (commerciallyavailable). When cultures reached turbidity equal to McFarlandnephelometer barium sulfate standard #1, a series of subcultures weremade by removing 0.1 ml aliquots and adding these to tubes containing 5ml of the same culture medium. Culture tubes were removed fromincubation at 1 to 2 day intervals and autoclaved at 250° F. for onehour. These tubes were then stored at -20° C. until assayed.

Nine fungi and ten bacterial species obtained from recent clinicalisolates were cultured to luxurient growth at 35° C. in Middlebrook 7H9medium. The organisms included the following: Candida albicans,Cryptococcus neoformans, Fusarium species, Aspergillus niger,Aspergillus fumigatus, Norcardia asteroides, Torulopsis glabrata, Mucorspecies, Syncephalostrum species, Pencillium species, Listeriamonocytogenes, Arizona hinshawii, Proteus mirabilis, Salmonella derby,Corynebacterium diptheriae, Providencia stuartii, Serratia marcescens,Yersinia enterocolitica, Streptococcus fecalis and Staphylococcusepidermidis. The cultures were autoclaved at 250° F. for one hour andstored frozen until assayed.

Sputum samples from patients with pulmonary disease were decontaminatedby 15 minute exposure to one volume of a solution containingN-acetyl-L-cysteine, 4% NaOH, and 2.9% Na citrate and culturedaerobically at 35° C. in Middlebrook 7H9 medium. Portions of thecultures were sampled between two and six weeks, autoclaved as describedabove and coded in the mycobacteriology laboratory prior to assay.

Results. A typical standare curve using the tracer-containing proteinhaving a molecular weight of 24,000 Daltons as the labeled antigen isshown in FIG. 4. The range of minimal detectability (about 5 ng/ml) andthe concentration required to produce half-maximal depression of the B/Fratio (about 25 ng/ml) are the same for autoclaved and unautoclaved PPD.The immunoreactivity in multiple dilutions of autoclaved Middlebrook 7H₉media containing M. tuberculosis is superimposable on this standardcurve.

The concentrations of immunoreactive PPD in sputum cultures of patientswith pulmonary infection are shown in Table 1:

                  TABLE I                                                         ______________________________________                                        Immunoreactive PPD Concentration in Sputum Culture                            from Patients with Pulmonary Infection.                                               PPD        Identification of Mycobacterium                            Patient (μg/ml)   by Culture                                               ______________________________________                                        1       2.9        M. tuberculosis                                            2       23.6       M. tuberculosis                                            3       1.6        M. intracellulare                                          4       26.4       M. tuberculosis                                            5       2.0        M. tuberculosis                                            6       4.8        M. tuberculosis                                            7       25.1       M. tuberculosis                                            8       1.0        M. intracellulare                                          9       29.8       M. tuberculosis                                            10      0.6        M. intracellulare                                          11      0.6        M. intracellulare                                          12      23.0       M. tuberculosis                                            13      0.8        M. fortuitum                                               14      0.8        M. scrofulaceum                                            15      1.2        M. intracellulare                                          16      1.1        M. scrofulaceum                                            17      0.3        M. simiae                                                  18       ND*                                                                  19      ND                                                                    20      ND                                                                    21      ND                                                                    22      ND                                                                    23      ND                                                                    24      ND                                                                    25      ND                                                                    26      ND                                                                    ______________________________________                                         *ND = not detectable                                                     

All cultures were maintained from 2-6 weeks. In no case wereMycobacteria identified by direct acid-fast smear of sputum.Immunoreactive tuberculoprotein was detected in all cultures in whichMycobacteria were identified. The concentrations were highest incultures of M. tuberculosis, ranging from 2.0 to 29.8 μg/ml. In thesecultures detectable immunoreactivity preceeded identification of M.tuberculosis by 14 to 28 days. The range of concentrations for speciesother than M. tuberculosis was from 0.3 to 1.6 μg/ml. No immunoreactivematerial was detected in cultures which failed to grow Mycobacteria.Sputum from patients 18-26 failed to grow after decontamination. Priorto decontamination, these sputa contained organisms which are amongthose listed above. Cultures of the organisms listed contained nodetectable immunoreactivity.

Table 2 shows the results of immunoassay of cultures of M. tuberculosisand M. fortuitum which were initiated as 1:50 dilutions of McFarland 1cultures.

                  TABLE 2                                                         ______________________________________                                        Concentration of Immunoreactive PPD in Cultures                               of M. Tuberculosis and Fortuitum.                                                         M. tuberculosis                                                                           M. fortuitum                                          Day         ng/ml       ng/ml                                                 ______________________________________                                        1           <100        <100                                                  2           120         <100                                                  4           580         <100                                                  5           820         <100                                                  6           1360        <100                                                  ______________________________________                                    

During six days of growth, there was a step-wise increase in theimmunoreactive PPD measured in cultures of M. tuberculosis while noimmonoreactive PPD was detected in cultures of M. fortuitum. Since thedoubling time of M. tuberculosis is shown to be approximately 24 hoursand the concentration of immunoreactive PPD in the culture medium alsodoubled at about this rate, the immunoreactivity is a direct measure ofthe number of organisms. This finding is confirmed by colony counts on7H10 plates. Antibiotic sensitivity can be determined by measuring theaccumulation of immunoreactivity in the presence and absence ofinhibiting concentratitons of antibiotics.

SUMMARY AND DISCUSSION

The data presented here indicate that the immunoreactive material beingdetected is quite specific for Mycobacteria. Although material derivedfrom M. tuberculosis reacts more strongly, there is cross-reactivitywith other Mycobacterial species. Immunoreactive materials were detectedin culture media of all Mycobacterial species studied. Noimmunoreactivity was detected in culture media from diverse bacterialand fungal species. The accumulation of immunoreactive material incultures is proportional to the numbers of organisms. The pattern ofcross-reactivity appears favorable since it allows identification ofMycobacterial disease caused by a variety of species. The method can beused for diagnosis and for determining the spectrum of antibioticsensitivity of Mycobacteria.

The obvious need for more rapid methods for the diagnosis oftuberculosis and other Mycobacterial infections brought about by thetechniques of the present invention are increased sensitivity, safety ofworking with autoclaved, non-infectious material and the ability toanalyze multiple samples rapidly and at low cost.

Having now fully described this invention, it will be apparent to one ofordinary skill in the art that the same can be carried out with minormodifications which do not effect the content or spirit thereof.

What is claimed as new and intended to be secured by Letters Patent ofthe United States is:
 1. A method for detecting the presence ofMycobacteria in a fluid or tissue which comprises:mixing said fluid ortissue containing a secretory product of Mycobacteria with a complex ofa tracer-containing molecule, and a binding macromolecule havingreversible binding affinity for said tracer-containing molecule, anddetecting said tracer containing molecule wherein said tracer-containingmolecule is a heat-stable charcoal-adsorbable labeled protein obtainedfrom the growth media of Mycobacterium tuberculosis, said protein havinga molecular weight of between 20,000 and 30,000, immunochemicalstability over a temperature range of 4° C.-250° C., a pH range of3.0-9.0 and exhibiting comigration towards the anode in the presence ofbovine serum albumin and borate buffer at a pH of 8.6 with an appliedvoltage of 80 volts on starch gel electrophoresis for 12 hours.
 2. Themethod of claim 1, wherein said binding macromolecule is an antibodyraised against secretory tubercular protein derived from a bacterium ofthe genus Mycobacterium.
 3. The method of claim 2, wherein saidbacterium is Mycobacterium tuberculosis.
 4. The method of claim 1,wherein said tracer-containing molecule is selected from the groupconsisting of a radiolabeled molecule, a chromophore labeled moleculeand an enzyme-labeled molecule.
 5. The method of claim 4, wherein saidtracer-containing molecule is a radiolabeled molecule labeled with ¹²⁵I.
 6. The method of claim 1, wherein said fluid is the biological fluidof a human or non-human animal.
 7. The method of claim 6, wherein saidfluid is the biological fluid of a human animal.
 8. The method of claim1, wherein said Mycobacteria being detected are selected from the groupconsisting of M. tuberculosis, M. intracellulare, M. scrofulaceum, M.simiae, M. bovis, M. leprae, M. avium, M. phlei, M. smegmatis, M. batty,M. kansasii, M. fortuitum, M. africanium, M. ulcerans, M. marinum and M.xenopi.
 9. The method of claim 8, wherein said Mycobacteria is M.tuberculosis.
 10. The method of claim 1, which further comprisesdetermining the susceptibility of Mycobacteria to antibiotic substancesby measuring the accumulation of secretory product of Mycobacteria whichare growing in the presence of said antibiotic substances.
 11. Themethod of claim 1, which, after mixing said complex with said fluid ortissue, further comprises:separating tracer-containing molecule which isbound on said binding macromolecule, from tracer-containing moleculewhich is free therefrom, and then determining the ratio of said bound tosaid free tracer-containing molecule.
 12. The method of claim 11,wherein said separation of said bound tracer-containing molecule fromsaid free tracer-containing molecule is carried out byabsorbing saidfree tracer-containing molecule on the surface of a material havingabsorption properties therefor.
 13. The method of claim 12, wherein saidmaterial is selected from the group consisting of charcoal, silicatesand hydroxyapatite.
 14. The method of claim 13, wherein said material ischarcoal.
 15. The method of claim 11, wherein said separation of saidbound tracer-containing molecule from said free tracer-containingmolecule is carried out byadding an antibody raised against said bindingmacromolecule and thereby precipitating any bound tracer-containingmolecule.
 16. The method of claim 15, wherein said antibody is attachedto an insoluble solid support.
 17. The method of claim 11, wherein saidseparation of said bound tracer-containing molecule from said freetracer-containing molecule is carried out by gel filtrationchromatography.
 18. The method of claim 11, wherein said separation ofsaid bound tracer-containing molecule from said free tracer-containingmolecule is carried out by fractional solvent precipitation.
 19. Themethod of claim 18, wherein said solvent is selected from the groupconsisting of ethanol, dioxane, polyethylene glycol, trichloroaceticacid, a solution of sodium sulfite, and a solution of ammonium sulfate.20. The method of claim 1, wherein said binding macromolecule isattached on an insoluble support.
 21. The method of claim 20, whereinsaid insoluble support is a particle of a cross-linked resin.
 22. Themethod of claim 21, wherein said resin is selected from the groupconsisting of dextran and cellulose.
 23. The method of claim 20, whereinsaid solid support is the interior of a test tube.
 24. The method ofclaim 20, wherein said solid support is a polyolefin disc.
 25. Themethod of claim 20, wherein said binding macromolecule is covalentlyattached to said solid support.
 26. The method of claim 20, which, aftermixing said complex with said fluid or tissue, furthercomprises:separating tracer-containing molecule which is bound on saidbinding macromolecule, from tracer-containing molecule which is freetherefrom, and then determining the ratio of said bound to said freetracer-containing molecule.
 27. The method of claim 1, wherein saiddetection of said tracer-containing molecule is carried out byscintillation counting.
 28. The method of claim 1, wherein saiddetection of said tracer-containing molecule is carried out by acolorimetric assay.
 29. The method of claim 1, which is used for thedetection of infectious tuberculosis in humans.